The invention is a process for the production of lipid-free, capsular polysaccharide from cultures of bacteria, including type-specific capsular polysaccharide from Gram-negative bacteria, free of endotoxin, without incurring substantial loss of the capsular polysaccharide. The process converts capsular polysaccharide which has covalently bound glycerol diester moieties into lipid free polysaccharide. In a preferred embodiment of the invention, lipopolysaccharides, LPS or endotoxin, is also removed from the capsular polysaccharide from which covalent lipid has been cleaved.
In one embodiment of the invention, the process is adapted to the production of lipid-free polyribosyl ribitol phosphate, hereinafter referred to as PRP, which is the capsular polysaccharide derived from cultures of the pathogenic bacterium Haemophilus influenzae type b, also referred to as Hib. The PRP produced is suitable as a component for the preparation of vaccines for induction of immune responses which protect against development of diseases caused by Hib. From this invention, it is predictable that the capsular polysaccharide of other pathogenic bacteria which have a similar covalent lipid, as in E. coli or Neisseria meningitidis, may be produced in similarly high yield by employing the teaching disclosed herein.
The pathogenic bacterium, Hib, is responsible for a number of diseases, the most significant of which is bacterial meningitis and systemic bacterial disease which occurs primarily in children under 5 years of age. Recently, vaccines directed against Hib have been licensed for use in humans by the FDA, see 1991 Physicians Desk Reference, pp. 1476-1478 for Merck & Co., Inc.'s PedvaxHIB.RTM., and pp. 1174 1176 for Lederle's Hib TITER.RTM..
One method of producing a vaccine against Hib is provided by the process described in U.S. Pat. Nos. 4,695,624 and 4,882,317. The PRP used in that process is derived from culturing Haemophilus influenzae type b, and isolating a fraction of polysaccharide from the culture medium. The isolated PRP is then conjugated with the outer membrane protein complex from Neisseria meningitidis b which acts as an immune enhacing carrier for the PRP which by itself is poorly immunogenic in infants.
Referring now to U.S. Pat. No. 4,695,624, an 800 L fermentation of Haemophilus influenzae type b is concentrated, in column 16, line 24, to 377 g of wet paste. The PRP is recovered by selective precipitation at different ethanol concentrations in the presence of calcium chloride. In column 17, line 1, 68 grams of dry product is obtained, hereinafter referred to as pre-phenol powder. Further workup involves phenol extraction and ethanol precipitation, yielding 39 grams of dry product in column 17, line 49, and then, finally ending up with 34.7 grams of dry product in column 18, line 14. This material will hereinafter be referred to as post-phenol powder. The post-phenol powder may be conjugated or subjected to further purification to remove endotoxin.
Endotoxin is a major contributor to elevation of temperature in mammals upon inoculation with bacterial derived immunogens. This so-called pyrogenic response may be eliminated by removal of lipid A containing lipopolysaccharides, hereinafter referred to synonomously as endotoxin, pyrogen or LPS, from the immunogen. In order to achieve this goal, the post-phenol PRP product obtained above may be further purified so that regulatory standards for pyrogen are met. Until the development of the instant invention, this was achieved by losing about 70% of the PRP present in the post-phenol powder, by selective ethanol fractionation. This involves precipitation of LPS from the post-phenol PRP by addition of ethanol to a sufficient concentration to just initiate precipitation, the so-called cloud point as outlined in U.S. Pat. Nos. 5,039,610 and 5,045,456. Ethanol is added to the cloud point, which is where about a two-fold increase in turbidity is achieved. An additional 0.5-2% ethanol is added, and the precipitate, which was heretofor a waste-product, hereinafter referred to as low-cut, is obtained, while lipid-free PRP remains in solution. Low-cut contains about 70% of the PRP and essentially all of the LPS.
In an attempt to overcome the loss of PRP which results from selective ethanol fractionation for endotoxin removal, the inventors of the process of U.S. Pat. No. 5,019,502 developed a method to remove LPS without substantial loss of PRP. The method involves passage of solubilized post-phenol powder through a hydrophobic adsorption step, either in the batch mode or column chromatographic mode, and preferably using nonionic resins to which endotoxin binds, but to which polysaccharides do not bind.
Use of a resin of highly porous styrene and divinylbenzene copolymer, such as HP20 does indeed quantitatively remove essentially all endotoxin, while providing almost quantitative yield of the PRP. This material is hereinafter referred to as post-HP20 PRP. However, a substantial portion of the PRP produced by Hib is in the form of a covalent lipo-PRP.
The possibility that PRP is produced by Haemophilus influenzae type b as lipo-PRP was first recognized by Kuo et al., [J. Bacteriol. 163, 769-773 (1985)], wherein Haemophilus influenzae type b was grown in a liquid medium supplemented with radioactive palmitate and/or radioactive ribose. The polyribosyl ribitol phosphate purified from the culture supernatant contained both radioactive ribose and palmitate which had been incorporated into the PRP during biosynthesis. Phospholipase A.sub.2 (PLA.sub.2) treatment was shown to remove some of the radioactive palmitate from the PRP whereas methods that would disrupt noncovalent association were unsuccessful. A structure for lipo-PRP was not given, but the data presented was consistent with the data reported in an earlier publication which proposed a structure for the meningicoccal group A, B, C and E Coli K92 polysaccharides [Gotschlich et al., J. Biol. Chem. 25b, 8915-8921 (1981)]. In the present invention, work with specific phospholipases has revealed a structure for lipo-PRP which is consistent with that presented in the detailed description below.
Lipo-PRP does not bind to the hydrophobic resin used in the U.S. Pat. No. 5,019,502 invention, due to the minor hydrophobic nature conferred on lipo-PRP by the covalent lipids, as opposed to the overwhelmingly anionic nature of the very large polysaccharide portion of the molecule. Thus, the final product obtained from that process contained a substantial portion of lipo-PRP as well as lipid-free PRP. This lipo PRP is removed, either by selective ethanol fractionation, which incurs about a 70% loss of PRP, or by employing the process deisclosed herein, whereby essentially all of the PRP is recovered as lipid-free PRP.
Thus, an object of this invention is to provide a process for recovery of lipo-free capsular polysaccharide such that a conjugate product is produced that is indistinguishable from conjugate prepared using only that fraction of lipid-free polysaccharide obtained by selective alcohol fractionation. Another object of this invention is to provide a process for high-yield production of PRP which comprises conversion of lipo-PRP into lipo-free PRP, rather than discarding the lipo-PRP. Another object is to provide a process which is reproducible for the amount and consistency of PRP obtainable from cultures of Haemophilus influenzae type b to ensure consistency of conjugate vaccine produced with the PRP so produced. Other advantages and objects of the invention will become apparent from the complete description as follows.